Tissues from the body taken for diagnosis of disease processes are often processed in the histology laboratory to produce thin tissue sections which can be mounted on slides and viewed under a microscope by a pathologist for analysis. These pre-analytical processes generally include, in order, gross examination fixation, dehydration, clearing, paraffin infiltration and embedding. The procedure is used for processing tissues including biopsies, larger specimens removed at surgery, or tissues from autopsy.
Gross examination generally consists of describing the macroscopic specimen and placing all or selected parts of it into a small plastic cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed into a fixative.
Following gross examination, a tissue is fixated. A purpose of fixation is to preserve tissues permanently in as life-like a state as possible by altering structures of proteins such that degradation by autolysis does not occur. A variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated. Major groups of fixatives, classified according to mechanism of action include aldehydes, mercurials, alcohols, oxidizing agents and picrates. Formalin fixation is best carried around neutral pH, for example, in the range of 6-8. Hypoxia of tissues tends to lower the pH, so there should be buffering capacity in the fixative to prevent excessive acidity. Common buffers include phosphate, bicarbonate, malate, cacodylate, and veronal. Commercial formalin, for example, may be buffered with phosphate at a pH of 7. Penetration of tissues depends upon the diffusability of each individual fixative. One way to improve penetration of a fixative is to gross (cut) the tissue thinly (2 to 3 millimeters (mm)). Penetration into a thin tissue section will occur more rapidly than for a thick section. The volume of fixative is generally important with a 10:1 ratio or greater of fixative to tissue typically targeted. Agitation of the specimen in a fixative will often also enhance fixation.
Once the tissue has been fixed or fixated, the tissue needs to be processed into a form in which it can be made into thin sections for microscopic examination. The usual way this is done is with paraffin. Tissues embedded in paraffin, which provides a solid support matrix for the tissue, allowing it be sectioned at a thickness on the order of 2 to 20 microns. Getting fixed tissue into paraffin for sectioning is called tissue processing with the main steps in this process being dehydration, clearing, infiltration and embedding.
Tissues fixed in aqueous solutions cannot be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. This may be done with a series of alcohols at different concentrations (e.g., 70 percent to 95 percent to 100 percent). Alternatively, the dehydration is done with a mixture of formalin and alcohol. Other dehydrants can also be used such as acetone or mixtures of different solvents.
Following dehydration, the tissue is cleared. “Clearing” consists of removal of the dehydrant and some of the lipids with a substance that will be miscible with the embedding medium (e.g., paraffin). The most common clearing agent is xylene.
Once cleared, the tissue is infiltrated with an embedding agent such as paraffin. Finally, the tissue in a cassette or removed from its cassette is placed into molten paraffin and then the paraffin is cooled to form a solidified block embedding or encapsulating the tissue so that it can be sectioned. Alternatively, the tissue can be processed in a sectionable cassette, embedded in paraffin along with the cassette and sectioned. Once the tissue has been embedded in a solid paraffin block, the tissue can be cut into sections that can be placed on a slide. This is done with a microtome. Once sections are cut, they are floated on a warm water bath that helps remove any wrinkles. The tissue sections in paraffin are then picked up from the water bath and placed on a glass microscope slide.